Protease inhibitors characterisation by SDS-PAGE and MALDI-TOF from Alocasia macrorrhizos and their modulation of macrophage immune-inflammatory properties.

This study describes the extraction and identification by electrophoretic and spectrometric techniques of protease inhibitor from the medicinal plant Alocasia macrorrhizos as well as investigates their immunomodulatory properties and cell viability. The A. macrorrhizos tubers were subjected to protease inhibitor extractions and characterised using SDS-PAGE and MALDI-TOF. The protein extracts were assessed for activities trypsin inhibition stoichiometry, haemagglutinating, cell viability, NO and TNF-α production inhibition. Concerning the protease inhibitors analysis through SDS-PAGE, the results showed two bands with 11 and 24 kDa, and the MS analysis detected the ions more intense of m/z 4276.795 and 8563.361 in the roasted protein extract. The IC50 of trypsin inhibition was 0.119 and 0.302 mg L-1 in the roasted and crude tuber, respectively. The protease inhibitors extract from the roasted tubers showed a reduction in the production of NO and TNF-α at concentrations lower than 100 µg mL-1, without a reduction in cell viability.

Metabolic profiling by LC-DAD-MS, FTIR, NMR and CE-UV of polyphenols with potential against skin pigmentation disorder.

In traditional Brazilian medicine, tubers extracts from Alocasia macrorrhizos are widely used in the treatment of skin pigmentation disorder. However, studies that evaluate its benefits in the treatment of this disorder are non-existent. Thus, this work aims to investigate the bioactivity of A. macrorrhizos extracts in cell culture and murine model of Vitiligo and correlating with its phenolic profile. The metabolic profiling from the bioactive extracts was obtained by LC-DAD-MS, FTIR, NMR, and CE-UV. The murine model of Vitiligo was induced with 5% hydroquinone in C57BL/6 male mice, which were treated or not with 100 mg/kg of roasted tuber aqueous extract. In Vitiligo model assay was observed hair follicle repigmentation and reduction of the epidermal layer thickness at the histopathological level, in the animals treated with aqueous extract of roasted tubers. The present study provides new molecular insight and scientific evidence on the potential utility of the extract of A. macrorrhizos against Vitiligo.

Alternative method for microenzymatic inhibition activities monitoring from Baccharis trimera extracts by CZE-UV.

INTRODUCTION: Capillary zone electrophoresis with direct UV detection (CZE-UV) was used to investigate the hypothesis about the extract of Baccharis trimera enzymatic activities as an analytical approach to monitoring the phenomenon. OBJECTIVE: The aim of this work was to investigate enzymatic bioactivities of the hydroalcoholic and infusion extracts of B. trimera through screening evaluation of the inhibition of the enzymes acetylcholinesterase (AChE) and α-glycosidase (α-GLY). METHOD: An alternative approach using CZE-UV to hydroalcoholic and infusion extracts of B. trimera monitoring was applied to evaluate the inhibition ability of the enzymes AChE and α-GLY. The result of the reaction of acetylthiocholine (AThCh) with AChE was thiocholine (TCh) and acetic acid, and from the amount of TCh generated, the AChE inhibition was calculated. For the inhibition study of the two enzymes, the reactions of the extracts were optimised to be performed in situ, inside the capillary column, and the introduction of the solutions was performed through ordered sequential plug injections. RESULTS: Samples extracted with 70% ethanol presented 7.80% inhibition for AChE and 0.51% for α-GLY, while samples extracted with 96% ethanol resulted in 6.89% inhibition for AChE and no inhibition activity for α-GLY. CONCLUSION: In the present work, the potentialities of CZE-UV for the study of hydroalcoholic and infusion extracts of B. trimera were demonstrated. The experimental results were useful for the calculation of the percentage of the inhibition activities of the AChE and α-GLY enzymes.

Mass spectrometry applied to diagnosis, prognosis, and therapeutic targets identification for the novel coronavirus SARS-CoV-2: A review.

Mass spectrometry (MS) has found numerous applications in medicine and has been widely used in the detection and characterization of biomolecules associated with viral infections such as COVID-19. COVID-19 is a multisystem disease and, therefore, the need arises to carry out a careful and conclusive assessment of the pathophysiological parameters involved in the infection, to develop an effective therapeutic approach, assess the prognosis of the disease, and especially the early diagnosis of the infected population. Thus, the urgent need for highly accurate methods of diagnosis and prognosis of this infection presents new challenges for the development of laboratory medicine, whose methods require sensitivity, speed, and accuracy of the techniques for analyzing the biological markers involved in the infection. In this context, MS stands out as a robust analytical tool, with high sensitivity and selectivity, accuracy, low turnaround time, and versatility for the analysis of biological samples. However, it has not yet been adopted as a frontline clinical laboratory technique. Therefore, this review explores the potential and trends of current MS methods and their contribution to the development of new strategies to COVID-19 diagnosis and prognosis and how this tool can assist in the discovery of new therapeutic targets, in addition, to comment what could be the future of MS in medicine.

Ensuring Homogeneity in Powder Mixtures for Pharmaceuticals and Dietary Supplements: Evaluation of a 3-Axis Mixing Equipment.

The mixing process plays a pivotal role in the quality of pharmaceuticals and food/dietary supplements, as it can impact the homogeneity of the substances in their dosage form and affect characteristics such as dissolution and stability. Thus, the choice of the right mixing device is paramount for compounding pharmacies. In this paper, we evaluated the mixing efficacy of a new 3-axis mixer device and determined its optimal working conditions. Three different formulations were compounded with the device and a total of 540 individual assays were performed by HPLC or ICP-MS to validate its use, in addition to a direct comparison among it and two alternative mixing methods. The 3-axis mixer device was able to provide homogeneous mixtures and finished capsules with adequate content uniformity with a broad range of conditions of use (mixing times from 2 to 8 min, and speed of rotation from 10 to 100 rpm). In addition, the device was superior to classical mixing methods (such as the use of manually shaken plastic bags) and at least equivalent to well-established ones (Y-shaped mixer). Finally, we proposed a cleaning procedure that was also adequate to prevent cross-contamination among products compounded with the same device.

Miconia albicans and Curcuma longa herbal medicines positively modulate joint pain, function and inflammation in patients with osteoarthritis: a clinical study.

This study aims to evaluate the analgesic and modulating effect of Curcuma longa and Miconia albicans herbal medicines in knee's osteoarthritis (OA) treatment. This longitudinal study evaluated 24 patients with OA. The patients were divided into three groups: ibuprofen (1200 mg/day), C. longa (1000 mg/day) and M. albicans (1000 mg/day). The medications were applied orally for 30 days. The synovial fluid of the knee joint was collect at the first (day 0) and the last medical (day 30) consultation. The groups treated with herbal medicines presented the same results when compared to Ibuprofen. The comparison of the means of Total WOMAC for M. albicans before and after treatment presented a statistically significant difference (mean day 0 = 57.19; mean day 30 = 31.02) as well as variation of Total WOMAC for C. longa (mean day 0 = 54.79; mean day 30 = 37.08). The WOMAC Total and the VASP were compared, it was found that there was a significant decrease in the means in the C. longa and M. albicans groups, as well as in the Ibuprofen group after treatment. The study demonstrated that the treatment of knee OA with C. longa or M. albicans positively interferes with patients pain and functionality, decreased WOMAC and VASP scores, leading to functional improvement of these patients. This is the first clinical study demonstrating the analgesic and anti-inflammatory effect on knee osteoarthritis from M. albicans comparable to Ibuprofen drug.

In vitro and in vivo evaluation of anti-inflammatory activity and free radical scavenging potential of leaves extract from Anadenanthera colubrina.

The present study highlights the antioxidant and anti-inflammatory activities from Anadenanthera colubrina leaves ethanolic extract (EEAc) and its phytochemical profile. The chemical profile of EEAc was determined and in vitro free radical scavenging potential, cell viability in RAW 264.7 and in vitro and in vivo anti-inflammatory activity were evaluated. The analysis of EEAc showed several phenolic compounds such as tannins and phenolic acids. The high antioxidant potential observed is possibly due to its high phenolic content. The EEAc (500 mg kg-1) showed an in vivo effect at the same level of dexamethasone; in vitro, at 50 µg mL-1, inhibited approximately 80% of nitric oxide production concentration, showed an inhibition of more than 50% of TNF-α production and presented high cell viability. The results show that A. colubrina leaves are an important source of phytochemicals that possesses antioxidant and anti-inflammatory properties.

Cecropia hololeuca: A new source of compounds with potential anti-inflammatory action.

Our objective is to investigate the phytochemical components, antioxidant capacity and in vitro and in vivo anti-inflammatory action from Cecropia hololeuca bark aqueous extract (AECh). The chemical characterization of AECh was performed through CE-UV, FTIR and NMR Spectroscopy. In vitro assays were performed with the AECh on murine macrophages J774A.1 cells in order to analyse cell viability, NO, TNF-α and IL-1ß productions and the in vivo anti-inflammatory potential in acute carrageenan paw oedema in mice. The AECh showed a decrease in the production of NO, TNF-α and IL-1ß, without altering the cell viability and reduction of the paw thickness in the 2nd, 3rd and 4th hour. The extract presented 72% free radical scavenging, 0.60% flavonoid content and showed the presence of gallic acid, caffeic acid and catechin as major constituents. The C. hololeuca bark extract showed important antioxidant and anti-inflammatory activity, emphasizing the industrial and pharmacological potential of this plant.

Structure and redox stability of [Au(III)(X

The choice of the auxiliary ligand in Au(III) complexes is of paramount importance in tuning their reactivity and biological activity. Tertiary phosphines are one of the most used auxiliary ligands in gold compounds, due to their stereo-electronic properties that confer stability and lipophilicity to these metallodrugs. The redox stability of [Au(III)(C^N^C)PR3]+ (A) (C^N^C = 2,6-diphenylpyridine) and [Au(III)(N^N^N)PR3]3+ (N^N^N = 2,2':6',2″-terpyridine) (B) complexes (where R is the phosphine substituent groups with different steric and electronic properties) was herein investigated for a set of 41 phosphines, using the predicted standard reduction potential (εo) for Au(III)/Au(I) electrochemical system as reference. For the complexes A, εo spread over 829 mV and all values were negative, whereas for the complexes B εo were positive and covered a narrower range of 507 mV. The phosphines with high buried volume (%Vbur ≥ 32%) decrease the complex stability despite being strong σ-donors. Both steric and electronic properties were used as molecular descriptors to build quantitative structure-property relationships (QSPR), which showed that the %Vbur plays the major role on the redox stability of the studied Au(III) complexes. For complexes B where the phosphine affects both Au(III) and Au(I) forms, the steric impact is more pronounced on the Au(I) reduced species. The electron-donating ability of phosphines is also important and plays a greater role on the redox stability of complexes B than complexes A. These outcomes are certainly useful to predict the redox stability of Au(III) complexes which, in turn, should affect their chemical reactivity against biological targets.

Baseline separation of α and ß-acids homologues and isomers in hop (Humulus lupulus L.) by CD-MEKC-UV.

An alternative method for simultaneous baseline separation of α and ß-acids homologues and isomers in hop by CD-MEKC with UV detection was proposed. The optimized background electrolyte was composed of 30 mmol/L sodium tetraborate solution, 45 mmol/L sodium dodecyl sulfate, 20 mmol/L ß-cyclodextrin and 10% v/v acetonitrile. The instrumental conditions were evaluated by using a 33 Box-Benhken experimental design. In order to demonstrate the applicability of the method, 21 hop samples from different varieties were analyzed. The repeatability intra- and interday tests were performed and relative standard deviations lower than 7% for area and migration times were observed. The present method comprehended 8 min analysis time and revealed to be faster and more efficient when compared to previous reports from literature.

Dual-opposite end multiple injection method applied to sequential determination of Na+, K+, Ca+2, Mg+2 ions and free and total glycerol in biodiesel by capillary zone electrophoresis.

A novel method for the sequential determination of sodium, potassium, calcium, and magnesium and free and total glycerol in biodiesel by capillary zone electrophoresis is proposed herein. The inorganic cations were separated along an effective length of 43.5 cm. The samples to quantify the free and total glycerol were injected into the opposite capillary end, close to the detection window, with an effective length of 8.5 cm. It was possible to achieve the separation of six analytes within 3 min. The quantification limits for the cations and glycerol ranged from 0.071 to 0.5 mg kg-1 to and 0.0017% to 0.017% w/w, respectively. Despite the complexity of the injection steps, the values for the instrumental, intraday and interday precision were better than 2.13, 4.49 and 5.68% (RSD), respectively, for the cations and the free and total glycerol. The method has good accuracy and specificity, which was statistically confirmed through an interlaboratory assay, where the method was compared with official methods.

Simultaneous determination of aspartame, cyclamate, saccharin and acesulfame-K in powder tabletop sweeteners by FT-Raman spectroscopy associated with the multivariate calibration: PLS, iPLS and siPLS models were compared.

For the first time, a procedure for simultaneous determination of the main artificial sweeteners, aspartame (ASP), cyclamate (CYC), saccharin (SAC), and acesulfame-K (ACSK) by a spectroscopic method associated with the multivariate calibration is proposed. These analytes were quantified in tabletop sweeteners samples using FT-Raman spectroscopy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used as reference method. Partial least squares (PLS), interval PLS (iPLS), and synergism PLS (siPLS) methods were evaluated in a comparative study where the selected interval models presented better results. Multivariate regression models, such as PLS, iPLS and siPLS were built and the lower root mean square errors for prediction (RMSEP) found were 0.027-0.031% w/w, 0.316-0.363% w/w, 0.082-0.184% w/w, and 0.040-0.049% w/w to ASP, CYC, SAC, and ACSK, respectively. The coefficient of determination for prediction (R2p) varied between 0.978 and 0.979, 0.969-0.977, 0.952-0.994, and 0.959-0.965 for ASP, CYC, SAC and ACSK, respectively. The analysis of model's residues was made by bias and permutation tests to evaluate systematic and trend errors. The selected intervals by iPLS and siPLS were evaluated and the bands related to the vibrational modes of the analytes were assigned with the aid of density functional theory calculations (DFT).

Simultaneous Determination of First-Line 4-FDC Antituberculosis Drugs by UHPLC-UV and HPLC-UV: A Comparative Study.

Tuberculosis is the second most deadly infectious disease, surpassed only by HIV/AIDS, and has resulted in over 1 billion deaths in the last 200 years. The World Health Organization estimates that in 2014, 9.6 million people were infected by this disease and 1.5 million had died. First-choice treatment consists of fixed-dose combination tablets containing rifampicin, isoniazid, pyrazinamide, and ethambutol hydrochloride (4-FDC). There are pharmacopeial protocols available to test 4-FDC, but they are prolonged, two-step methods. One single-step method in the literature performs the simultaneous determination by HPLC, but requires a long acquisition time. In this context, an ultra-HPLC (UHPLC) method was developed based on the HPLC method with the objective of reducing analysis time. A C18 column (1.9 µm particle size) was used with UV-diode-array detection at 238 and 282 nm. The method was found to be selective, linear, exact, precise, and robust. Samples from two batches were analyzed and the results compared with those obtained by the HPLC method, with no statistically significant differences observed (P > 0.05). This UHPLC method reduced the analysis time from 17 to 4 min, with a more than 90% reduction in sample and reagent consumption and a financial economy of almost 50-fold.

Simultaneous Determination of First-Line 4-FDC Antituberculosis Drugs by UHPLC-UV and HPLC-UV: A Comparative Study.

Tuberculosis is the second most deadly infectiousdisease, surpassed only by HIV/AIDS, and hasresulted in over 1 billion deaths in the last200 years. The World Health Organization estimatesthat in 2014, 9.6 million people were infected bythis disease and 1.5 million had died. First-choicetreatment consists of fixed-dose combinationtablets containing rifampicin, isoniazid,pyrazinamide, and ethambutol hydrochloride(4-FDC). There are pharmacopeial protocolsavailable to test 4-FDC, but they are prolonged,two-step methods. One single-step method in theliterature performs the simultaneous determinationby HPLC, but requires a long acquisition time.In this context, an ultra-HPLC (UHPLC) methodwas developed based on the HPLC method withthe objective of reducing analysis time.A C18 column (1.9 µm particle size) was used withUV-diode-array detection at 238 and 282 nm. Themethod was found to be selective, linear, exact,precise, and robust. Samples from two batcheswere analyzed and the results compared with thoseobtained by the HPLC method, with no statisticallysignificant differences observed (<em>P</em> > 0.05). ThisUHPLC method reduced the analysis time from17 to 4 min, with a more than 90% reduction insample and reagent consumption and a financialeconomy of almost 50-fold.

Optimization of Ultrasound-Assisted Extraction of Cr, Cu, Zn, Cd, and Pb from Sediment, Followed by FAAS and GFAAS Analysis.

An ultrasound method for simultaneous extraction of Cr, Cu, Zn, Cd, and Pb from sediment, and determination by flame atomic absorption spectrometry (FAAS) and graphite furnace atomic absorption spectrometry (GFAAS) was proposed. The experimental results obtained using analytical curves and the method of standard additions agreed at a confidence level of 95% for all the analytes, as determined by FAAS and GFAAS, indicating no significant matrix effects. Recoveries ranged from 80.1 to 93.7% (certified reference material) and from 89 to 107% (spike tests). The LOD and LOQ results from the method were consistent with the techniques used (FAAS and GFAAS), with high analytical throughput. The proposed method was then used to determine Cr, Cu, Zn, Cd, and Pb in river sediment samples from Rio Doce, Minas Gerais, Brazil. The results indicated levels below those permitted by Brazilian legislation for all the analytes, with the exception of Cr.

A rapid method for total Β-escin analysis in dry, hydroalcoholic and hydroglycolic extracts of Aesculus hippocastanum L. by capillary zone electrophoresis.

INTRODUCTION: Seeds of Aesculus hippocastanum L. are used in European phytotherapy to treat inflammatory and vascular problems, and also to help in the regulation of the microcirculation. Thus, the quality control of herbal medicines using this species is important. OBJECTIVE: To develop and to optimise a capillary zone electrophoresis method to determine total ß-escin in different extracts of A. hippocastanum L. METHODS: The optimal condition found through chemometric approach was: 25 mmol/L of bicarbonate-carbonate buffer, pH 10.3; +20 kV of voltage; 20°C of cartridge temperature; direct ultraviolet detection at 226 nm; 13 mbar injection for 5 s and analysis time within 6 min. RESULTS: Repeatability, coefficient of variation (CV; %) = 3.19, 3.07 and 1.89 (n = 12), and intermediate precision, CV (%) = 3.05, 3.53 and 2.99 (n = 24) for dry, hydroalcoholic and hydroglycolic extracts, respectively were achieved. The accuracy was evaluated through recovery tests in concentration levels of 100, 150 and 200 g/L, ranging from 98.17 to 104.68%. The proposed method exhibited linearity (r = 0.9983) in the concentration range from 101.4 to 907.2 g/L and limits of detection and quantification equal to 11.63 and 38.76 g/L respectively. CONCLUSION: A fast and reliable methodology for determination of total ß-escin was successfully validated and applied on extracts of A. hippocastanum L. demonstrating its usefulness to quality control of medicines containing this plant species.

Simultaneous determination of first-line anti-tuberculosis drugs by capillary zone electrophoresis using direct UV detection.

An alternative methodology for simultaneous analysis of ethambutol, isoniazid, rifampicin and pyrazinamide in pharmaceutical formulations by capillary zone electrophoresis under UV direct detection with an analysis time of 8.0 min is proposed. Background running was based on the effective mobility curve of the analytes and an optimum separation condition was achieved using a 3(3) Box-Behnken design, with Brij 35, Cu(2+) and acetic acid/sodium acetate buffer as factors. An electrolyte consisting of 50.0 mmol L(-1) of acetic acid/sodium acetate buffer, 12.5 mmol L(-1) of CuSO(4), and standard and sample solutions prepared in 2.00 mmol L(-1) of Brij 35 and 12.5 mmol L(-1) of CuSO(4) were optimized. After evaluating validation parameters, the method was successfully applied to the analysis of samples in the form of tablets and sachets.

Optimization of an electrolyte system for analysis of ethambutol in pharmaceutical formulations by capillary zone electrophoresis using complexation with copper(II).

An alternative methodology for the determination of ethambutol by capillary zone electrophoresis (CZE) under direct UV detection at 262 nm, using acetic acid/sodium acetate buffer solution (pH 4.6) containing copper(II) sulphate to form the ethambutol-copper(II) complex, within analysis time of 2.5 min is proposed. The optimum CE conditions for the background electrolyte were established performing experiments of a 3(2) factorial design. Complex formation was evidenced by the UV batochromic shift and the [CuETB](0) and [CuETB](2+) chemical structures were indicated by LC-MS analysis. After some validation parameters have been performed, such as linearity (r=0.999), selectivity (comparison between slope of the calibration curve of the external standard and calibration curve of the standard addition), area precision (RSD%: <2.13 for ETB and <1.94 for 2A1B), recovery mean (101.7% for ETB and 99.95% for 2A1B) and quantification limit (mg L(-1): 10.17 for ETB and 19.70 for 2A1B), the method was successfully applied to ETB analysis in pharmaceutical formulation samples. It is possible to determine the presence of the 2A1B impurity at concentrations of less than 1% ETB content.

Determination of olive oil acidity by CE.

In this work, a CE method for the determination of olive oil acidity was proposed. The method was based on an ethanolic extraction (at 60 degrees C) of the oil long-chain free fatty acids (LC-FFAs) components followed by CE determination in pH 6.86 phosphate buffer at 15 mmol/L concentration containing 4 mmol/L sodium dodecylbenzenesulfonate (SDBS), 10 mmol/L polyoxyethylene 23 lauryl ether (Brij 35), 2% v/v 1-octanol and 45% v/v ACN under indirect UV detection at 224 nm. Although this electrolyte promoted baseline separation of myristic acid (C14:0) (internal standard (IS)) and olive oil major components (palmitic acid (C16:0), oleic acid (C18:1c) and linoleic acid (C18:2cc)) in less than 8 min, after a few injections, the electropherogram profiles were severely altered (peak broadening, migration time shifts, etc.) and the current increased substantially. An adsorption study was conducted revealing that the dissolution of the capillary external polyimide coating during the electrophoretic run caused the detrimental effect. After removal of the capillary tip coating, ten consecutive injections could be performed without any disturbances and this simple procedure was, therefore, implemented during quantitative purposes. The reliability of the proposed method was further investigated by the determination of acidity of an extra virgin olive oil sample in comparison to the established methodology (AOCS method Ca 5a-40, alkaline volumetric titration (AVT)). No statistical differences were found within 95% confidence level. A % acidity of 0.39 +/- 0.02 was found for the olive oil sample under consideration.

Simultaneous separation of five fluoroquinolone antibiotics by capillary zone electrophoresis.

A novel methodology has been developed for simultaneous separation of ciprofloxacin (CPFLX), gatifloxacin (GTFLX), levofloxacin (LVFLX), moxifloxacin (MFLX) and sparfloxacin (SPFLX) fluoroquinolone antibiotics (FQs), using capillary zone electrophoresis (CZE) with UV detection at 282 nm. Electrolyte composition was optimized through the variation of the Tris/hydrochloride and sodium tetraborate buffer mixture. The electrolyte consisted of a 25 mmol L(-1) Tris/hydrochloride and 15 mmol L(-1) sodium tetraborate buffer mixture resulting in pH 8.87. All analytes were separated in less than 3 min. The proposed method was applied to the separation of FQs in pharmaceutical formulations, and the assay results were within 95-105% of the label claim.